Microbial extracellular enzyme activity is responsible for much of the carbon and nutrient cycling in freshwaters, and logically there should a relationship between the chemical properties of a system and its enzymatic profile. To evaluate this concept, we surveyed extracellular enzyme activity in five large rivers (the Upper Mississippi, Missouri, Ohio, Tennessee, and Arkansas) of the Mississippi River Basin, one of the world’s largest river systems. The rivers drain areas of different climate, physiography, and land use, and differ in their physicochemical properties. Despite differences in nutrient concentrations, there were no consistent differences in enzyme activity between the five rivers, with as much variation in activity between sites on the same river as among different rivers. Enzymatic profiles were dominated by leucine aminopeptidase, phosphatase, and β-glucosidase, and appreciable enzymatic activity was still present following the removal of particles (3-micron filtration) or cells (0.22-micron filtration). The proportion of particle- or cell- associated enzymatic activity contributing to overall activity varied between enzymes, being higher for β-glucosidase, leucine aminopeptidase, and N-acetyl-β-D-glucosaminidase than for sulfatase or cellobiohydrolase. Dissolved elemental stoichiometry suggested that bacterioplankton in all rivers were limited by C overall, with P also being more limiting than N. While regional-scale patterns in enzyme activity in large rivers may indicate anthropogenic influences, this study demonstrates that finer-scale variation, such as between sites on the same river, or between particles and free-living bacterioplankton, may be equally as important.